1. Field of the Invention
The present invention relates to a flow cytometry apparatus for determining one or more characteristics of particles passing therethrough, and more particularly, concerns a flow cytometry apparatus with improved optical features.
2. Description of the Prior Art
There are a number of cell or particle analyzing devices employing flow cytometry techniques which rely on hydrodynamically focused fluid flow through a passageway for determining specific characteristics of the flowing cells or particles. Flow analysis of particles has been employed in the determination of a variety of characteristics of individual particles. This analysis is most useful in analyzing or determining characteristics of cells for the collection of information which would be useful in areas of research, hematology, immunology and the like. The researcher, for instance, may be interested in determining specific characteristics of individual cells so that such cells may be classified, identified, quantified, and perhaps sorted for further investigations or analysis.
Three instruments which rely on hydrodynamically focused fluid flow systems are sold by Becton, Dickinson and Company. One device, known as the ULTRA-FLO 100.TM. Whole Blood Platelet Counter, rapidly and reliably counts whole blood platelets in the hematology laboratory. In the ULTRA-FLO 100.TM. system, a trajectory of a diluted sample containing platelets passes straight through the center of the counting chamber orifice since the sample fluid is focused by a sheath of pressurized fluid. Another instrument sold by Becton, Dickinson and Company, relying on a hydrodynamically focused fluid flow system is known as the FACS.TM. analyzer. The FACS.TM. analyzer rapidly analyzes cells on the basis of fluorescence and electronic volume properties. Analysis is accomplished by introducing cells in suspension to the center of a focused liquid stream and causing the cells to pass, substantially one at a time, through the filtered and focused light from a high-power mercury-arc lamp. Each cell is individually characterized by its electronic impedance volume and by the intensity and color of fluorescence emitted while it is illuminated. Another instrument known as the FACS.TM. sorter utilizes fluid flow principles which are similar to the FACS.TM. analyzer, but further sorts the cells based on specifically detected characteristics. In all of the aforementioned systems, a sheath fluid is utilized to focus the particles or cells as they pass through the passageway associated with the analyzing or counting capabilities. Further, the FACS.sup.TM analyzer employs an optically clear or transparent liquid flow chamber, sometimes referred to as a flow cell, through which a stream of cells passes. Light is directed orthogonally through this flow cell to intercept the particles in a focal region thereof. Scattered light or fluorescence emitted by the particles may be detected to provide information with respect to each passing particle. U.S. Pat. Nos. 4,348,107; 4,240,029; 4,165,484 and 4,110,604 describe particle analysis sytems in which particles flowing in a stream are enveloped in a sheath fluid which focuses and confines the sample fluid (with particles) to the center of the flowing stream.
In flow cytometry apparatuses in which an incident beam of light is relied upon for obtaining information with respect to the particles, one or more lenses are normally involved in focusing the light on the particles flowing within the particle stream. Such lenses are also relied upon to collect light emitted by or scattered from the particles. One such lens assembly embodied within a particle analyzer instrument, and utilizing a transparent liquid flow chamber, is described in copending, commonly assigned patent application Ser. No. 276,738, filed in the U.S. Pat. and Trademark Office on June 24, 1981, and entitled, "Analyzer for Simultaneously Determining Volume and Light Emission Characteristics of Particles." In the invention of the aforementioned patent application, the lens assembly is positioned adjacent the outer surface of the transparent liquid flow chamber, with a thin layer of glycerol at the interface between lens assembly and flow chamber. This glycerol is an index matching medium provided to facilitate light transmission and minimize light losses. Even with this arrangement between the lens assembly and the flow chamber, a rather complicated alignment procedure is typically required to bring the particles flowing in the liquid stream through the flow chamber into the focal plane of the collection lens. For example, a three-axis adjustable lens mount is provided to establish the relative axial position of the lens assembly and flow chamber. The stability of such a lens mount is an area which needs improvement. Moreover, there is no mechanism in the presently known and used flow cytometry apparatuses to adjust the position of the flowing particle stream in order to provide the final focus with respect to the light passing through the collection lens. Of course, it is known to provide vernier adjustments for aperture sizes and microscope adjustments in flow cytometry apparatuses. For example, such vernier adjustments are described in U.S. Pat. Nos. 3,675,768 and 3,924,947.
Most present-day commercial flow cytometry apparatuses employ flow cells or chambers having cylindrical orifices and sample particle streams having a circular cross-section. Due to this geometry, significant optical aberrations, which limit the efficiency of both light collections and excitation (related to fluorescently labeled particles), are present. Moreover, these aberrations increase geometrically as the numerical aperture of the lens is increased. In general, the higher the numerical aperture, the higher the sensitivity of the flow cytometry apparatus. Aberrations thus serve to limit the practicality of using high numerical aperture lenses. In addition, as sample particle flow rate is increased, the diameter of the sample stream is increased, requiring a lens of increased depth of focus. Depth of focus is inversely proportional to the lens numerical aperture, and hence large depth of focus and high numerical aperture are mutually exclusive. It has been recognized that a large rectangular orifice within the transparent liquid flow chamber would be beneficial in optimizing the light transmission into or out of the transparent liquid flow cell. Such square orifices are described in Thomas, R.A., et al., "Combined Optical and Electronic Analysis of Cells with the AMAC Transducers," The Journal of Histochemistry and Cytochemistry, volume 25, number 7, pages 827-835, 1977, and in U.S. Pat. No. 4,348,107. It was pointed out in U.S. Pat. No. 4,348,107, however, that the optical and mechanical characteristics of a particle analyzer using a square sensing orifice enclosed inside a cube formed by adhering four pyramids together has proven to be suboptimal.
Accordingly, it is evident that improvements in the optical elements and features of flow cytometry apparatuses are still being sought which would improve the efficiency, accuracy and dependability of the light transmission characteristics related to such flow cytometry apparatuses. It is to such improvements that the present invention is directed.